Molecular recognition touches virtually every aspect of biomedical research, from the discovery of new therapeutic leads that selectively modulate a target's function to the development of new diagnostic tools. One- bead-one-compound (OBOC) combinatorial library synthesis and screening has the potential to source and discover such ligands cheaply and efficiently. However, the process is plagued by the selection of false positive beads displaying ligands that do not recapitulate their tethered binding behavior once liberated from the bead surface. All combinatorial library screening must therefore incorporate hit re-synthesis, purification, and solution-phase binding assays for validation, making the investigation of false positives resource intensive to the point of negating the efficiency of the combinatorial library synthesis and screening. Directly screening each library member for solution-phase target binding would alleviate the burdens described above, but such technology does not yet exist. This proposal will explore polarization-activated droplet sorting (PADS), which will be capable of performing ultra-miniaturized picoliter-scale solution-phase fluorescence polarization (FP) assays on each library member during primary screening. DNA-encoded combinatorial library beads will be prepared with fluorescently-labeled ligand linked to the bead via photocleavable linker, distributed into target- containing microfluidic droplets, photochemically cleaved from the bead in flow, assayed in droplets for binding by laser-induced FP, and sorted if the FP threshold is exceeded. Validation of PADS will involve examining various positive control authentic ligands and their known targets (e.g., streptavidin/biotin, pepstatin A/HIV-1 protease) and proceed to a known case of a false positive discovered in the development of a screen for CCR5-mimetic ligands of the HIV-1 gp120/CD4-Ig complex. PADS will be assessed for its ability to differentiate the authentic ligand from the known false positive in these droplet-scale solution-phase binding assays. The successful development of PADS will allow screening of diverse (>105 members) small molecule OBOC libraries for authentic binding, requiring negligible amounts of target. PADS will integrate the efficiency advantages of DNA-encoded combinatorial library synthesis and miniaturized screening to provide a highly scalable platform for the discovery of new molecular tools and therapeutic leads.